G protein-coupled receptors comprise the largest family of eukaryotic signal transduction proteins that communicate across the membrane. We report the crystal structure of a human β 2 -adrenergic receptor-T4 lysozyme fusion protein bound to the partial inverse agonist carazolol at 2.4 Å resolution. The structure provides a high-resolution view of a human G protein-coupled receptor bound to a diffusible ligand. Ligand-binding site accessibility is enabled by the second extracellular loop which is held out of the binding cavity by a pair of closely spaced disulfide bridges and a short helical segment within the loop. Cholesterol, a necessary component for crystallization, mediates an intriguing parallel association of receptor molecules in the crystal lattice. Although the location of carazolol in the β 2 -adrenergic receptor is very similar to that of retinal in rhodopsin, structural differences in the ligand binding site and other regions highlight the challenges in using rhodopsin as a template model for this large receptor family.* To whom correspondence should be addressed: stevens@scripps.edu; kobilka@stanford.edu $ These authors contributed equally Author Contributions: RCS and BKK independently pushed the GPCR structural biology projects for more than 15 years. BKK managed the protein design, production and purification. RCS managed novel crystallization and data collection methods development and experiments. VC developed novel methods for, and performed LCP crystallization, LCP crystal mounting, LCP data collection, model refinement, analyzed the results, and was involved in manuscript preparation. DMR supplied protein materials for all crystallization trials, grew and collected data from the bicelle crystals, collected, processed and refined the 3.5 Å LCP structure, refined the 2.4 Å structure, analyzed the results, and was involved in manuscript preparation. MAH designed the blind crystal screening protocol and collected the 2.4 Å data set, processed the 2.4 Å data, solved the structure by MR at 3.5 Å and 2.4 Å resolution, wrote the initial draft of the manuscript and created all figures. SGFR assisted with the final stages of β 2 AR-T4L purification. FST expressed β 2 AR-T4L in insect cells and, together with TSK, performed the initial stage of β 2 AR purification. HJC assisted with the refinement. PK assisted in developing novel methods to screen the transparent crystals, data collection, refinement, and was involved in manuscript preparation. WIW assisted with low resolution data collection and processing, solved the β 2 AR-T4L molecular replacement problem at 3.5 Å, participated in the 2.4 Å refinement process, and participated in structure analysis and manuscript preparation. BKK additionally assisted with β 2 AR-T4L purification, β 2 AR-T4L 3.5 Å synchrotron data collection, structure analysis and manuscript preparation. BKK and DMR designed the β 2 AR-T4L fusion protein strategy. RCS additionally assisted with β 2 AR-T4L crystallization, 2.4 Å data collection, structure solution, refinem...

Chemokine receptors are critical regulators of cell migration in the context of immune surveillance, inflammation and development. The G protein-coupled chemokine receptor, CXCR4, is specifically implicated in cancer metastasis and HIV-1 infection. Here we report five independent crystal structures of CXCR4 bound to an antagonist small molecule IT1t and a cyclic peptide CVX15 at 2.5–3.2 Å resolution. All structures reveal a consistent homodimer with an interface involving helices V and VI that may be involved in regulating signaling. The location and shape of the ligand binding sites differ from other G protein-coupled receptors and are closer to the extracellular surface. These structures provide new clues about the interactions between CXCR4 and its natural ligand CXCL12 and with the HIV-1 glycoprotein gp120.

The role of cholesterol in eukaryotic membrane protein function has been attributed primarily to an influence on membrane fluidity and curvature. We present the 2.8 A resolution crystal structure of a thermally stabilized human beta(2)-adrenergic receptor bound to cholesterol and the partial inverse agonist timolol. The receptors pack as monomers in an antiparallel association with two distinct cholesterol molecules bound per receptor, but not in the packing interface, thereby indicating a structurally relevant cholesterol-binding site between helices I, II, III, and IV. Thermal stability analysis using isothermal denaturation confirms that a cholesterol analog significantly enhances the stability of the receptor. A consensus motif is defined that predicts cholesterol binding for 44% of human class A receptors, suggesting that specific sterol binding is important to the structure and stability of other G protein-coupled receptors, and that this site may provide a target for therapeutic discovery.

Topoisomerases I promote the relaxation of DNA superhelical tension by introducing a transient single-stranded break in duplex DNA and are vital for the processes of replication, transcription, and recombination. The crystal structures at 2.1 and 2.5 angstrom resolution of reconstituted human topoisomerase I comprising the core and carboxyl-terminal domains in covalent and noncovalent complexes with 22-base pair DNA duplexes reveal an enzyme that "clamps" around essentially B-form DNA. The core domain and the first eight residues of the carboxyl-terminal domain of the enzyme, including the active-site nucleophile tyrosine-723, share significant structural similarity with the bacteriophage family of DNA integrases. A binding mode for the anticancer drug camptothecin is proposed on the basis of chemical and biochemical information combined with these three-dimensional structures of topoisomerase I-DNA complexes.